Microbial contamination of transplant tissues and blood transfusion products is a major medical problem. Blood banks are faced with a great challenge in testing each platelet bag for microbial contamination prior to release for infusion into a patient. Part of this challenge relates to the relatively short shelf-life of platelet samples, typically 5-7 days and sometimes shorter. The challenge with respect to platelets is further complicated by standard storage conditions. Platelets, unlike most other transplantable tissues, do not tolerate refrigeration and disappear rapidly from the circulation of recipients if subjected to even very short periods of chilling. This cooling effect on platelet survival is thought to be irreversible and renders platelets unsuitable for transfusion. When platelets are exposed to temperatures lower than 20° C., they rapidly undergo modifications in shape indicative of impairment. The need to keep platelets at room temperature (e.g. 22-25 C) prior to transfusion has imposed a unique set of costly and complex logistical requirements for platelet storage. Because platelets are metabolically active at room temperature, they are typically subjected to constant agitation in gas permeable containers to allow for the exchange of gases to prevent the toxic consequences of metabolic acidosis. These storage conditions encourage the growth of bacteria thereby creating a higher risk of bacterial infection. Because screening methods that rely on detection by culture can take longer than the usable shelf-life to detect contamination, contaminated platelets are often infused into patients, and the physician is notified subsequently that the platelets were contaminated as the culture results become available. Under the American Association of Blood Banks (A.A.B.B.) standard 5.1.5.1, blood banks or transfusion services are instructed to have methods to limit and detect bacterial contamination in all platelet concentrates. Nevertheless, the risk of transfusion with bacterially contaminated platelets may be as a high as 1 in 1,000 units, with perhaps to 25 of such incidents resulting in adverse effects on patients. While some contamination may derive from donor bacteraemia, contamination at the time of collection by bacteria present on the skin or in blood packs are main sources of contamination that cannot be addressed through donor diagnostic screening.
Common methods to mitigate contamination include preventative measures (e.g. arm cleansing, diverting a first portion of collected blood, and filtration) and culture methodologies. As evidenced by the current rate of contamination, these procedures remain inadequate. Moreover, because culture-based detection methods require significant incubation periods (sometimes days), samples may not be used for a significant portion of their useful life, and when they are used, detection may not be complete. This is particularly true for contamination by bacteria that are relatively slow growing. For example, the mean detection times for Bacillus sp. Staphylococcal sp. Streptococcal sp. Micrococcus luteus Kocuria varians Corynebacterium sp. and Propionibacterium sp. have been estimated as 24 hours, 27 hours, 34 hours, 47 hours, 56 hours, 87 hours, and 97 hours, respectively, using standard culture methods.
One commercially available test is referred to as the BacT/ALERT test (bioMérieux, Inc., Durham, N.C.). Bacterial detection is based on the evolution of carbon dioxide by proliferating bacteria. A carbon-dioxide-sensitive liquid emulsion sensor at the bottom of the culture bottle changes color and is detected through alteration of light reflected on the sensor. Another method for bacterial detection involves measuring the oxygen content in a platelet preparation sample. An example is the Pall eBDS test (Pall Corporation, Port Washington, N.Y.). The approach to detection measures the oxygen content of air within the sample pouch as a surrogate marker for bacteria. An oxygen analyzer is used to measure the percent of oxygen in the headspace gas of the pouch or bag having the platelets. If bacteria are present in the platelet sample collected, an increasing amount of oxygen is consumed through the metabolic activity and proliferation of the bacteria in the sample during incubation, resulting in a measurable decrease in oxygen content of the plasma as well as the air within the sample pouch. While the non-specific measure of bacterial growth permits detection of many kinds of bacteria, the sensitivity is relatively low and requires long incubation times.
Alternative methods to the standard culture-based screens include bacterial antigen detection and nucleic acid-based screening. A major limitation on antigen-based methods is that they cannot be applied directly for testing of samples where the spectrum of bacterial pathogens is unknown. Detection of common bacterial nucleic acid sequences, such as 16S rRNA has been proposed to achieve a broader spectrum of detection, but such methods have so far been deemed less sensitive and specific than current culture methods and not appropriate for early testing of platelet samples.